Analytic detection of biomolecules, e.g., proteins, nucleic acids, and the like, is fundamental to molecular biology. In many applications, it is desirable to detect the presence of one or more particular molecules in a sample. For example, identification of a particular DNA sequence within a mixture of restriction fragments is used to determine the presence, position, and number of copies of a gene in the genome. It is also an integral technique in DNA typing. Analytic detection is also used, e.g., in disease diagnosis and drug development, to determine the presence of a particular antibody or protein, e.g., in a blood sample or large chemical library. Detection of biomolecules is therefore of fundamental value in, e.g., diagnostic medicine, archaeology, anthropology and modem criminal investigation. To meet these needs many techniques, e.g., DNA blotting, RNA blotting, protein blotting, and ELISA assays, have been developed to detect the presence of a particular molecule or fragment in the midst of a complex sample containing similar molecules.
For example, western blotting is useful for detecting one or more specific proteins in a complex protein mixture, such as a cell extract. The procedure involves fractionating the protein mixture, generally by denaturing polyacrylamide gel electrophoresis, and transferring and immobilizing the mixture onto a solid membrane of either nitrocellulose or nylon by electroblotting. The loaded membrane is then incubated with an antibody raised against the protein of interest. The antibody-antigen complex so formed on the membrane is then detected by a procedure that typically involves the application of a second antibody, raised against the first antibody, and to which an enzyme has been covalently linked. The insoluble reaction product generated by the enzyme action can then be used to indicate the position of the target protein on the membrane. The sensitivity of detection can be increased by amplification of the signal using either the biotin-streptavidin system or by chemiluminescence detection.
This classical procedure is very time consuming and labor intensive. For example, transferring the proteins to a membrane is generally a time consuming step and is typically done by capillary blotting or by the faster and more efficient methods of vacuum blotting or electrophoretic blotting.
More recently, new and faster microfluidic methods of performing biological assays in microfluidic systems have been developed, such as those described by the pioneering applications of Parce et al., xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d WO 98/00231 and in Knapp et al., xe2x80x9cClosed Loop Biochemical Analyzersxe2x80x9d (WO 98/45481; PCT/US98/06723). For example, high throughput methods for analyzing biological reagents, including proteins, are described in these applications.
Improved methods for performing western blot and affinity assays are, accordingly desirable, particularly those which take advantage of high-throughput, low cost microfluidic systems. The present invention provides these and other features by providing high throughput microscale systems for analyte detection, western blots, and the like, and many other features that will be apparent upon complete review of the following disclosure.
The present invention provides methods, devices, systems, and kits for detecting a component of interest in a complex mixture. Typically, the method comprises separating a mixture of components, which mixture of components contains the component of interest. To detect the component of interest, the mixture of components or the separated components are contacted with a component-binding moiety specific to the component of interest. The component-binding moiety binds to the component of interest and is detected, thereby detecting the component of interest.
In one embodiment, the component of interest and the various components of the mixture are labeled with two detectably different labels so that both the component of interest and the mixture of components are concurrently detected.
In another embodiment, the separated components are bound to or adsorbed to a particle set. The particle set is optionally stacked in a detection region and a component-binding moiety specific to the component of interest is directed into the region of the device containing the particle set with the bound components. The component-binding moiety thereby binds to the component of interest, thus providing detection of the component of interest.
The devices, systems, and methods of the invention are useful in a variety of detection systems, e.g., western assays, biotin-avidin systems, lectin/carbohydrate systems, and in other applications that will be apparent upon further review.
In one aspect, the method comprises providing a body structure having a plurality of microscale channels disposed therein, the plurality comprising a microfluidic separation channel and at least one side channel intersecting the separation channel, wherein the separation channel and the side channel are fluidly coupled. A mixture of components is flowed through the separation channel, resulting in separated components. A labeled component-binding moiety is then flowed through a side channel and into the separation channel, wherein it binds to the component of interest. The component-binding moiety is then detected, thereby detecting the component of interest.
The separated components are typically labeled components that are optionally detected simultaneously with the component-binding moiety. This embodiment optionally includes deconvoluting the detection signal to identify the separated components and the component of interest. This embodiment includes two detectably different label moieties having detectably different spectral characteristics, such as different excitation or emission maximum. The different labels include, but are not limited to fluorescent labels, chemiluminescent labels and colorimetric labels. For example, the separated components are optionally labeled with a first fluorescent dye and the component-binding moiety is labeled with a second fluorescent dye. These two dyes are typically detectably different. In another embodiment, the component of interest and the component-binding moiety are optionally labeled with detectably different colorimetric labels. In another embodiment, the component of interest is labeled with one type of label, e.g., chemiluminescent, and the component-binding moiety is labeled with a second type of label, e.g., fluorescent.
In another aspect, a microfluidic system comprising a particle set is provided. A body structure having at least one microfluidic channel disposed therein is provided, and a mixture of components is flowed through the microfluidic channel, separating the mixture of components and producing separated components. The separated components are then bound to a particle set comprising a plurality of particle member types. The separated components bound to the particle set are then contacted with a component-binding moiety specific to the component of interest, thereby binding the moiety to the component of interest. The component-binding moiety is then detected, thus detecting the component of interest. After being bound to the separated components, the particle set is flowed into a detection channel downstream of the separation and binding events. The particle set is optionally stacked or fixed in the detection channel. In one embodiment, stacking occurs against a barrier located in the detection channel.
The particle set is comprised of a plurality of particle member types, that optionally comprise a polymeric material, a silica material, a ceramic-material, a glass material, a magnetic material, a metallic material, an organic material, or a combination of these materials. In one embodiment, binding comprises adsorbing the separated components onto the members of the particle set. In these embodiments, the particle member types optionally comprise PVDF, nitrocellulose, or a polyamide, such as nylon and the like.
In other embodiments, the particle set is contacted with a blocking solution after binding the separated components to the particle set and prior to contacting the particle set with the component-binding moiety, thereby binding blocking moieties to open sites on the particle set. The blocking moiety is optionally a blocking protein or buffer containing casein, solubilized non-fat dry milk, gelatin, or bovine serum albumin.
The particle set with the bound component of interest is typically incubated with the component-binding moiety for a time ranging from about 10 seconds to about 30 minutes.
In some embodiments, the method comprises washing the particle set or the bound complexes comprising a component-of interest and a component-binding moiety prior to detection, thereby substantially removing component-binding moieties that are not bound to the component of interest.
The component of interest in the above methods is optionally a protein, a carbohydrate, biotin, avidin, or the like. The component-binding moiety is optionally a protein-binding moiety such as an antibody or a carbohydrate-binding moiety, such as a lectin. The antibody or other binding moiety is preferably specific to the protein or other component of interest. In other embodiments, the component of interest optionally comprises biotin and the component binding moiety is avidin or the component of interest comprises avidin and the binding moiety is biotin.
The mixtures of components of the invention are separated in one embodiment by electrophoresis in a polymer or gel, such as a polyacrylamide solution, matrix, or gel. In some embodiments, the mixture of components is separated and concurrently bound to the particle set and in others the mixture is separated and contacted with the particle set after separation. In this case, separation is performed in a separation matrix and binding of the separated components to the particle occurs downstream of the separation matrix. In alternate embodiments, the components are contacted by the component-binding moiety during the separation or just after and then directed into a detection region where they are simultaneously detected. In this embodiment, the components are separated on the basis of molecular weight, which is then determined by the retention time. The separated components are optionally labeled with a fluorescent dye and detected upon elution from the separation channel.
Detection optionally comprises optically detecting a chemiluminescent, calorimetric, or fluorescent label moiety that has been fixed to the component-binding moiety. The detection channel is typically located within the at least one microfluidic channel or intersecting the at least one microfluidic channel; and, optionally comprises a stacked particle set proximal to a detector.
In other embodiments, the body structure comprises a detection channel fluidly coupled to the separation channel and the side channel. In these embodiments, the mixture of components is separated by flowing the mixture through a separation matrix located in the separation channel; wherein the component-binding moiety is flowed into the separation channel downstream of the separation matrix and upstream of a detection point proximal to the detection channel. The side channel in some embodiments is proximal to the detection region.
In another embodiment, the mixture of components is flowed through the separation channel concurrently with flowing the component-binding moiety into the separation channel. The component-binding moiety is flowed in the same direction or the opposite direction as the mixture of components. In some embodiments, the component-binding moiety has an electrokinetic mobility opposite to that of the mixture of components.
In other embodiments, the method further comprises washing the side channel and separation channel, thereby substantially removing component-binding moieties that are not bound to the component of interest. Furthermore, the signal from the labeled component-binding moiety bound to the component of interest is typically detectable above a background level.
In another aspect, the invention also provides microfluidic devices for detecting the components of interest. In one embodiment, a microfluidic device for detecting a component of interest is provided. The device comprises a plurality of fluidly coupled microscale channels disposed therein. The plurality of channels typically comprises a first channel, a second channel, a third channel, a binding region, a detection region, a stacking region and a particle set. The first channel comprises a component separation region in which a mixture of components is separated. The second channel intersects the first channel and comprises a particle set disposed therein, which particle set comprises a plurality of particle member types. The third channel, which comprises a blocking solution and a labeled component-binding moiety specific to the component of interest, intersects the first channel. The binding region is fluidly coupled to the first channel, for binding the mixture of components to the particle set. The detection region is fluidly coupled to the first channel; and, the stacking region is positioned within the detection channel.
A second embodiment of the device is also provided. This device is also used in detecting a component of interest but does not include a particle set. The device comprises a plurality of fluidly coupled microscale channels disposed therein. The plurality of channels comprises a main channel, a side channel, and a detection region. The main channel comprises a component separation region in which a mixture of components is separated. The side channel, which intersects the main channel, comprises a component-binding moiety, and the detection region is fluidly coupled to the main channel.
The particle sets and separation channels for the devices are typically the same as those discussed above. In addition, these devices are incorporated into integrated systems. The integrated systems comprise one of the microfluidic devices as described above as well as a fluid direction system, and a detection system.
A fluid direction system is fluidly coupled to the microfluidic device and transports the sample or components through the microscale channels. The sample and components useful in the integrated systems of the invention are the same components useful in the above-described methods. The fluid direction system in some embodiments is an electrokinetic based fluid direction system or in other embodiments, a pressure based system.
The detection system is positioned proximal to the detection region or detection channel and detects one or more of the component-binding-moiety, the separated components and the component of interest. The detection system comprises a detector that is optionally one of the following: a chemiluminescent detector, a fluorescent detector or a calorimetric detector.
The control system is operably linked to the fluid direction system and instructs the fluid direction system to deliver or transport the sample and/or components through the microfluidic channels. The control system in some embodiments comprises a computer and software.
The computer is operably linked to the integrated system and includes software. The software analyzes and deconvolutes signals produced from detection and directs fluid movement in the system. The software directs movement of one or more of the following: movement of a sample through the component separation region or channel, resulting in separated components; movement of a particle set and the separated components to a binding region, resulting in binding of the separated components to the plurality of particle member types; movement of the component-binding moiety to the binding region, resulting in binding of the component-binding moiety to the component of interest; and, movement of the particle set, separated components, and the component-binding moiety to the detection region, where the component-binding moiety is detected, thereby detecting the component of interest. In addition the software directs movement of the one or more of the following through the separation channel or binding region: a buffer solution, a blocking solution, and a washing solution. It also operates to direct the particle set to a stacking region in some embodiments.